Screen printing tissue models

ABSTRACT

A process of simply, cheaply, and reproducibly creating complex tissue models using screen printing and the tissue model prepared using the screen printing process. These models are amenable to high throughput screening. They will allow the study of components of disease progression and can be used for screening therapies.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is filed under the provisions of 35 U.S.C. § 111(a) andclaims priority to U.S. Provisional Patent Application No. 62/599,977filed on Dec. 18, 2017 in the name of Erin Lavik et al. and entitled“Screen Printing Tissue,” which is hereby incorporated by referenceherein in its entirety.

FIELD

The present invention relates to a process of simply, cheaply, andreproducibly creating complex tissue models using screen printing andthe tissue model prepared using said screen printing process. Thesemodels are amenable to high throughput screening. They will allow thestudy of components of disease progression and can be used for screeningtherapies. Being able to efficiently model tissues for high throughputscreening will allow for the investigation of the complex interactionsbetween the cells and structures involved, and, ultimately, will providea platform for investigating the interplay between the tissue and themicrobiome. Further, the process and tissue models can be used forimplantable therapies.

BACKGROUND OF THE INVENTION

High-throughput screening methods amenable to three dimensional culturesopens the possibility of efficiently investigating disease models andtherapeutic interventions. However, the ability to build threedimensional models for these high-throughput systems has been primarilybased on 3D printing, photolithography, and bioprinting. Each allows oneto develop patterns and architectures that are seen in vivo, but theyrequire materials and processes that are not always compatible withcells and progenitors, for example the shearing forces associated withmany of the 3D printing technologies and UV light for thephotopolymerizable approaches.

3D printing offers an exciting approach to facilitate organizing thecell types into models, for example, retinal models, but it is notwithout limitations particularly in relation to stem cells and retinalcells. Bioprinting allows printing of cells and hydrogels into complexarchitectures (Jung et al., 2016; Yue et al., 2016), whichadvantageously allows one to develop the kinds of patterns andarchitectures that are seen in vivo, but disadvantageously requiresextruding the materials and cells through fine openings with high shear,as well as specialized equipment. While the cost of bioprinters has comedown, the extrusion process requires bioinks that are compatible withthe shearing associated with the approach as well as materials thatprotect cells during this process (Dubbin et al., 2016).

There is a continued need to develop new approaches to make in vitrocomplex tissue models that are simple, reproducible, cost effective, andavoid the extrusion issues associated with bioprinting and the UV lightsource in photolithography.

SUMMARY OF THE INVENTION

The present invention relates to a process of screen printingmultilamellar structures that promote specific cellular organization andthe tissue model obtained using said process. These models are amenableto high throughput screening and they will allow the study of componentsof disease progression and have the potential to be used for screeningtherapies.

In one aspect, a synthetic multilamellar tissue model is described, saidmodel comprising (i) a substrate, (ii) a foundation comprising at leastone layer comprising a hydrogel, and (iii) at least one non-foundationallayer comprising one or more of proteins, cells, a hydrogel, a secondconstituent, collagen, and any combination thereof, wherein thesynthetic multilamellar tissue model comprises at least one patternhaving resolution in a range from about 20 μm to about 500 μm.

In another aspect, a process of making a multilamellar tissue model isdescribed, said process comprising:

(a) positioning a first screen having an exposed first pattern over asubstrate;

(b) placing a first solution to be printed onto the first screen,wherein the first solution comprises a hydrogel and optionally a secondconstituent;

(c) pushing a blade across the first screen to spread the first solutioninto the exposed first pattern;

(d) removing the first screen to reveal a first layer comprising thehydrogel and optionally the second constituent;

(e) positioning a second screen having an exposed second pattern overthe first layer;

(f) placing a second solution to be printed onto the second screen,wherein the second solution comprises one or more of proteins, cells,additional hydrogel, a second constituent, collagen, gelatin, and anycombination thereof;

(g) pushing a blade across the second screen to spread the secondsolution into the exposed second pattern; and

(h) removing the second screen to reveal a second layer positioned onthe first layer comprising the hydrogel,

wherein the process does not require exposure to of the layers UV light,bioinks, or shearing processes.

In yet another aspect, a synthetic multilamellar tissue model producedusing the process described herein is disclosed.

In still another aspect, a three-dimensional tissue model is described,said model comprising (a) the multilamellar tissue model of claim 1, and(b) layers positioned using bioprinting, photolithography, and/or 3Dprinting.

Other aspects, features and embodiments of the invention will be morefully apparent from the ensuing disclosure and appended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic of the process of screen printing.

FIG. 2 is an image of SOX2-GFAP(+)ON-aNPCS expressed in a double mutant(ER2-SOX2-Cre X Td-Tomato-LoxP(GFP) animal). Both GFP(+)/GFAP(+) aNPCsand GFAP(+) astrocytes are seen.

FIG. 3A is an image of early aNPC neurospheres grown from rodent ON.Scale bar: 50 um.

FIG. 3B is an image of aNPC neurospheres grown from rodent ON that weresubsequently grown on a laminin coated plate. Scale bar: 100 uM.

FIG. 4A is a scanning electron micrograph of decellularized ON. Thecells express microvillae and are SOX2(+)/GFAP(+)/Chx10(+) at thisstage.

FIG. 4B is a scanning electron micrograph of human ON-aNPCs extending onan ON-laminar surface.

FIG. 4C is another scanning electron micrograph of human ON-aNPCsextending on an ON-laminar surface.

FIG. 5A is an image of the growth of Retinal Ganglion Cells (RGCs) inPLL-based gels. RGCs cells migrate into hydrogels and readily extendelaborate neurites in three dimensions. RGCs were seeded onto hydrogelsand stained using calcein-AM (green) to label cell somas and neurites.Live confocal z-stack images were acquired. The RGCs retained theirstereotyped morphology.

FIG. 5B is an image of the growth of Amacrine Cells (ACs) in PLL-basedgels. ACs cells migrate into hydrogels and readily extend elaborateneurites in three dimensions. ACs were seeded onto hydrogels and stainedusing calcein-AM (green) to label cell somas and neurites. Live confocalz-stack images were acquired. The ACs retained their stereotypedmorphology.

FIG. 5C is an image of the growth of Retinal Ganglion Cells (RGCs) andAmacrine Cells (ACs) in PLL-based gels. RGCs and ACs were seeded ontohydrogels and stained using calcein-AM (green) to label cell somas andneurites. Live confocal z-stack images were acquired. The coculture ofRGCs and ACs retained their stereotyped morphology.

FIG. 5D is an image of retinal progenitor cells oriented in thepolarized fashion in response to being in polymer scaffolds (Lavik etal., 2005).

FIG. 5E is an image of retinal progenitor cells oriented in thepolarized fashion in response to being in PLGA scaffolds (Lavik et al.,2005). The cells follow architectural cues, and in doing so, they takeon morphologies that are consistent with retinal cells.

FIG. 6A is an image of a PLL-based gel labeled with FITC, which reactswith the charged free amines on lysine.

FIG. 6B is an image of anti-fibronectin immunostaining of PLL-based gelreacted with fibronectin. The fibronectin absorbs to the charged amineson the gels.

FIG. 7 is an illustration and image of a dye-loaded hydrogel printed ona hydrogel layer. The barbell structure is at the limits of theresolution available with a 100 μm mesh screen.

FIG. 8 is a schematic of the screen printing process for printingBruch's membrane and two of the cellular layers of a retinal tissuemodel. In this schematic, the green gel is cell-permissive, and theyellow is the non cell-permissive PEG-based gel.

FIG. 9 is a schematic of the screen printing process for printing acolon model.

DETAILED DESCRIPTION, AND PREFERRED EMBODIMENTS THEREOF

The present invention relates to a complex tissue model and a process ofmaking same, said process using screen printing, which avoids thedisadvantages associated with UV light, bioinks, and shearing describedhereinabove. The process is simple, reproducible, and highly scalable,making the tissue model suitable for high-throughput assays. Usingscreen printing, a range of gels and cells can be printed in multiplelayers and in complex patterns with high resolution and reproducibility.Preferably, the complex tissue model mimics a tissue that ismultilamellar in nature.

The changes in the extracellular matrix, particularly associated withBruch's membrane, play a significant role in the pathology of diseasesincluding age-related macular degeneration (AMD) but the methods toinvestigate this have been limited up until now (Al Gwairi et al., 2016;Booij et al., 2010; Hollyfield et al., 2008). By patterning a Bruch'smembrane structure and then building a retinal model, a healthy retinawith tight retinal pigment epithelium (RPE) junctions can be modeled aswell as the diseased retina with the degenerating RPE layer. By couplingthis approach with human RPE cells and human adult neural stem cellsderived from the eye and optic nerve, which have been shown to expressmarkers for the major retinal cell types, a tissue system can beproduced that models the 3D retina and optic nerve structures in ascalable and reproducible manner that is exceptionally well suited tohigh-throughput screening approaches for understanding and treatingdiseases of the retina. The process described herein allows for therecapitulation of the layers of the retina and provides the matrix cuesto promote the critical polarization of the cell types and promote theformation of appropriate synapses in the system, as well as the enhancedsurvival of target neurons.

The usefulness of the presently described tissue model and process ofproducing same is not limited to the modeling of retinal tissue.Neurodegenerative diseases are one of the greatest health burdens ofthis century, and yet the disease models are lacking (Schweiger et al.,2016, Tunesi et al., 2016, Choi et al. 2014). The critical cell typesassociated with neural models are particularly fragile, and viabilitycan be poor when they experience shear, especially through extrusionsystems (Kim et al., 2015, Gu et al., 2016). Thus, most work has focusedon printing materials to which the cells are added separately (Lozano etal., 2015) or the use of gels to reduce the shear experienced by neuralcells (Madl et al., 2015). Screen printing represents an alternativethat reduces the shearing and, based on our preliminary experiments,leads to robust survival of even extremely sensitive human iPS (inducedpluripotent stem) cells.

In addition, the prevalence of food allergies are growing withapproximately 7% of children and 6% of adults being diagnosed with oneor more food allergies including celiac disease (Sicherer et al., 2014).Beyond these conditions, diseases of the colon include irritable bowelsyndrome (IBS), a poorly understood but highly pervasive conditionaffecting approximately 11% of the population around the world (Canavanet al., 2014), and colon cancer. Approximately 4.4% of the populationwill be diagnosed with colon cancer in their lifetimes (Siegel et al.,2016). We need better models to understand these conditions and toscreen for therapies. Prior art in vitro intestinal models areintriguing systems but are not ideal for high throughput screening andtransport analysis due to their enclosed lumen limiting access to apicalcell surfaces. Advantageously, the intestine is well suited to screenprinting. The tissue is multilamellar, but to be functional, the cellsmust be organized appropriately. The resolution of the criticalarchitecture in the colon, the crypt, is 80-100 um in diameter (Araki etal., 1995) which is well within the range for screen printing describedherein. One of the most critical assays for the intestine is testing ofthe barrier function of the cultured cells, which is typicallyaccomplished by employing transepithelial electrical resistance (TEER)measurement (Srinivasan et al., 2015). We can build the electrodesrequired for TEER measurement into the screen-printed system usingstandard electrical screen printing techniques that are biologicallycompatible (Kadimisetty et al., 2016).

Taking a cue from the electronics industry (Peng et al., 2016; Suikkolaet al., 2016), the process described herein uses screen printing todevelop highly scalable hydrogel-cell tissue models. Screen printingoffers several advantages including, but not limited to, (1) modelingtissue comprising many types of materials and cells including those thatare sensitive to UV light and extrusion, (2) the materials and cells canbe patterned within a wide range of three-dimensional, multilamellarstructures, (3) by varying the screen structure, layers can be createdacross a wide range of thicknesses, and (4) these models can be builtquickly and at low cost. For example, a new tissue can be built within aday with screening materials for about $1 USD per finished pattern.

An example of the screen printing process is shown in FIG. 1. In screenprinting, an emulsion is applied to a screen and exposed to light tocure the emulsion. Printed transparencies having a pattern thereon arethen used as a mask to expose portions of the emulsion to UV light,followed by the removal of the unexposed areas, i.e., the pattern. Theprepared screen is then positioned over a surface (e.g., the substrate),solutions comprising gel precursors, cells, and/or other layercomponents are added to the screen, and a blade is used to move thesolutions over the screen to the removed pattern. Following removal ofthe screen, the pattern can be seen on the surface. For the biologicalscreen printing described herein, preferably the screens have pores in arange from about 20 μm to about 500 μm, more preferably about 80 μm toabout 500 μm. Other preferable resolutions include, but are not limitedto, about 80 μm to about 250 μm, about 80 μm to about 200 μm, about 80μm to about 150 μm, about 80 μm to about 120 μm, or about 250 μm toabout 500 μm. It should be appreciated that the screen for each newlayer may have the same pore size of, or a different pore size from, thescreen used for the previous layer, as readily determined by the personskilled in the art. In other words, each new layer may have the sameresolution, or a different resolution, from the previous layer.Preferably, the screen pore size chosen provides for maximumreproducibility and resolution and does not compromise the layercomponents, e.g., a substantial amount of undifferentiated cells survivepost-printing. For the purposes of this application, a “substantialamount of undifferentiated cells” corresponds to preferably greater than60%, more preferably greater than 70%, and even more preferably greaterthan 80% of the cells survive the screening process withoutdifferentiation and without the presence of a matrix which augmentssurvival.

As defined herein, the “substrate” can be any material that is inert tothe gels and/or cells printed thereon and can be sterilized including,but not limited to, glass, stainless steel, metals, ceramics, plastics,gas-permeable membranes such as a silicone (polydimethylsiloxane)membrane, and other polysiloxanes, fabric, degradable polymer films ormembranes, other polymer membranes, electrospun materials, and anycombination thereof. For the purposes of the present disclosure,reference will be made to the substrate being a glass slide but itshould be understood that it is not limited as such.

As defined herein, a “layer” corresponds to a material that is printedand that has a thickness that is substantially consistent over asurface, for example a surface that is substantially planar. It shouldbe appreciated that because of the nature of screen printing, a layercould have cavities, vias, holes, or some pattern therein having adifferent chemical and/or biological makeup than the rest of the layer.

As defined herein, “substantially devoid” corresponds to the presence ofless than 1 ppm, preferably less than 1 ppb, even more preferably lessthan 1 ppt of the particular material referred to.

As a foundation for the described process, hydrogels based onpoly(ethylene glycol) (PEG) can be synthesized, wherein the hydrogelsgel via vinylsulfone-thiol chemistry in 3-5 minutes (Jeong et al.,2014). In one embodiment, a hydrogel comprising PEG and polylysine (PLL)is synthesized, wherein the PLL-based gels are made by functionalizingthe PLL-PEG macromers with vinylsulfone and reacting with PEG-thiolmacromers to make the gels. By tuning the ratio of PEG with polylysine awide range of moduli can be achieved (Hynes et al., 2009; Zustiak etal., 2010). For example, by choosing the ratio of the two components andthe molecular weight, the elasticity of the resulting gels can becontrolled (Hynes et al., 2009, Zustiak & Leach, 2010, Zustiak et al.,2010, Royce Hynes et al., 2007). Other hydrogel foundations arecontemplated herein including, but not limited to, poly(ethylene glycol)coupled with at least one second constituent such as PLL, hyaluronicacid (HA), poly-γ-(glutamic acid) (γ-PGA), poly(aspartic acid) (PAA),and/or poly(arginine). Another hydrogel foundation comprises gelatin,with or without one or both of PEG or a second constituent. It should beappreciated by the person skilled in the art that any hydrogel materialthat gels in about 3-10 minutes can be used as the hydrogel foundation,wherein the choice is generally dependent on the cell type and thetissue being modeled. HA-based gels can be made in a similar manner tothe PLL-based ones with macromers of HA functionalized with PEG-thiolgroups combined with PEG-vinylsulfone macromers to make the hydrogels(Jeong et al., 2014). The presence of the second constituent, e.g., PLL,enables the absorption of proteins of interest into the gels,considerably reducing time and cost compared to using each protein as abasis for the gel (Hynes et al., 2009).

As defined herein, the “foundation” is the hydrogel-containing layer orlayers that is printed onto the substrate and serves as the platform forthe tissue model. The foundation supports the survival, organization,and maturation of the cells in this model system. By screen printing thehydrogel-containing layer or layers, i.e., the foundation, the physicalguidance cues can be provided through the cell permissive regions andnon cell-permissive regions as well as through the chemical guidancecues provided by the absorption of extracellular matrix molecules. Noncell-permissive regions can be made using a hydrogel consisting of orconsisting essentially of PEG. For example, the screen printed hydrogelfoundation can have architectural cues comprising columns of aPEG-polylysine matrix in a non cell-permissive matrix consisting of orconsisting essentially of PEG. The foundation can comprise one, two,three, four, five, six, seven, eight, nine, or ten or more layers,wherein each layer can be the same as or different from one another.Optionally, at least one layer of the foundation can comprise at leastone protein or at least one cell, as readily understood by the personskilled in the art.

As introduced hereinabove, the presence of the second constituent, e.g.,PLL, enables the absorption or reaction of proteins of interest to thegels. For example, FIG. 6A shows the PEG-PLL gel with absorbedfibronectin and FIG. 6B shows the PEG-PLL gel with absorbedrhodamine-antifibronectin antibody. The extracellular matrix proteinsabsorb or react with to the charged polylysine backbone of the gels. Theabsorption process is rapid and stable and the proteins do not releasefrom the gel over weeks (Hynes et al., 2009). In contrast, no proteinsabsorb to hydrogels consisting of or consisting essentially of PEG,i.e., non cell-permissive portions, which will impede cell attachment(Leach et al., 2004).

In practice, the screen having the specific pattern thereon is placedover the substrate, a solution comprising the components to be printedin the specific layers are combined, mixed together and placed over thesubstrate, and then a blade is pushed across the screen to ensure thatthe solution is evenly spread over the screen. The pattern is dependenton the layer being printed, as readily determined by the person skilledin the art. The hydrogel components to be printed are present at theappropriate concentration, for example in a range from about 1% to about20%, more preferably about 10% in solution, at the appropriate pH, forexample, physiological pH, wherein the concentration and pH aredependent on the material and the mechanics desired since the mechanicsof the resulting gel impact cell behavior. The solutions are preferablyaqueous and can include at least one of physiologically appropriatebuffers, salts, sugars, water, and other physiologically appropriatespecies including, but not limited to, proteins and drugs. A protein ofinterest can be absorbed to or reacted with the secondary constituentbefore printing the macromer, or the protein of interest can be printedon the gel in a separate step. The latter is appropriate when thecellular components interact with the surface. The positioning of theprotein within the gel (i.e., absorbed to or reacted with the secondaryconstituent) or on the gel is dependent on the resolution to beobtained, the effect on the robustness of lamination between layers, andthe desired extent of interaction of the cellular components with thesurface.

The thickness of each layer printed using the process described hereinwill be a function of the distance the screen is placed from thesubstrate or last layer printed, as readily determined by the personskilled in the art. The thickness of each layer can be in a range fromabout 5 μm to about 5 mm, depending on the desired tissue model. Spacerssuch as Mylar film (from 5 μm-400 μm) can optionally be used to set thedistance for one or more layers. Mylar is advantageous since it can beautoclaved and disposed of after printing to avoid contamination. Thethickness of each layer can be confirmed using any number of techniquesincluding, but not limited to, ellipsometry, atomic force microscopy(AFM), and scanning electron microscopy (SEM). Ellipsometry is suitablefor thicknesses less than 50 rpm. For greater thicknesses, SEM and AFMpreferred.

Because the process described herein uses vinylsulfone-thiol chemistry,the lamination between the gel layers is robust. The extent oflamination, or lack thereof, can be characterized using protocolspreviously developed in our laboratory involving a pull test (Sarkar etal., 2008) as well as the parallel plate rheometry for the laminatedstructures versus bulk material. In the event that the layers are notproperly laminated, a chemical bonding layer utilizing some of theamines in the hydrogel can be added (Id.).

The desired elastic modulus is dependent on the cells comprised therein.For example, preferably the hydrogels have an elastic modulus for neuralcells in a range from about 1000-8000 Pa, preferably in a range fromabout 3500-6000 Pa, for neural cell migration and orientation in and onthe hydrogel. Fibrin-containing gels preferably have a modulus in arange from about 100-10000 Pa (Wufsus et al., 2015). Gels comprisingvalvular interstitial cells preferably have a modulus in a range fromabout 100-6000 Pa (Rosales et al., 2015). Gels comprising thechondrogenic cell line, ATDC5, preferably have a modulus in a range fromabout 100-500 Pa, preferably about 150-250 Pa (Maeda et al., 2014). Gelscomprising human mesenchymal stem cells preferably have a modulus in arange from about 15 to about 75 Pa (Li et al., 2014). Gels comprisingSchwann cells preferably have a modulus in a range from about 4 to about12 kPa (Gu et al., 2012). Both the gelation time and the mechanicalproperties of each layer can be determined using a parallel platerheometer.

The gel components are synthesized and preferably filtered prior toprinting, e.g., through 0.45 μm filters, e.g., TEFLON™ filters. Thesolutions comprising the respective components to be printed are alsopreferably exposed to UV light in a tissue culture hood as a secondsterilization step. The screens, blades, and all the other materialsused in the process can be autoclaved.

Once the foundation is prepared, one or more non-foundational layers arepositioned thereon to complete the tissue model, wherein thenon-foundational layers comprise one or more of proteins, cells, (e.g.,epithelial cells, goblet cells, dendritic cells, neural cells, RPEcells, photoreceptor cells, bipolar cells, amacrine cells, horizontalcells), additional hydrogel as described herein, a hydrogel comprising asecond constituent as described herein, collagen, gelatin, drugs, andany combination thereof. The non-foundational layers can be positionedusing the screen printing process described herein, or by culturing, bystamping, and/or by absorption. For example, a solution comprising thecomponents of the specific non-foundational layer can be introduced to ascreen for printing. The solutions are preferably aqueous and comprisethe components of the specific non-foundational layer can include atleast one of physiologically appropriate buffers, salts, sugars, water,serum, and other physiologically appropriate species including, but notlimited to, proteins and drugs. It should be appreciated that there canbe one, two, three, four, five, six, seven, eight, nine, or ten or morenon-foundational layers, wherein each non-foundational layer can be thesame as or different from each other non-foundational layer, as readilyunderstood by the person skilled in the art.

Accordingly, in a first aspect, a synthetic multilamellar tissue modelis described, said model comprising (i) a substrate, (ii) a foundationcomprising at least one layer comprising a hydrogel, and (iii) at leastone non-foundational layer comprising one or more of proteins, cells, ahydrogel, a second constituent, collagen, and any combination thereof,wherein the synthetic multilamellar tissue model comprises at least onepattern having resolution in a range from about 20 μm to about 500 μm.Advantageously, the hydrogel sets without using UV radiation and thefoundation and non-foundational layer are both substantially devoid ofbioinks. Further, when the non-foundational layer comprises cells,greater than 60%, preferably greater than 70%, and more preferablygreater than 80% of the cells survive the screening process withoutdifferentiation and without the presence of a matrix which augmentssurvival.

In a second aspect, a process of making a multilamellar tissue model isdescribed, said process comprising:

(a) positioning a first screen having an exposed first pattern over asubstrate;

(b) placing a first solution to be printed onto the first screen,wherein the first solution comprises a hydrogel and optionally a secondconstituent;

(c) pushing a blade across the first screen to spread the first solutioninto the exposed first pattern; (d) removing the first screen to reveala first layer comprising the hydrogel and optionally the secondconstituent;

(e) positioning a second screen having an exposed second pattern overthe first layer;

(f) placing a second solution to be printed onto the second screen,wherein the second solution comprises one or more of proteins, cells,additional hydrogel, a second constituent, collagen, gelatin, and anycombination thereof;

(g) pushing a blade across the second screen to spread the secondsolution into the exposed second pattern; and

(h) removing the second screen to reveal a second layer positioned onthe first layer comprising the hydrogel,

wherein the process does not require exposure to of the layers UV light,bioinks, or shearing processes. Additional layers can be layered insuccession by positioning an nth screen having an exposed nth patternover the n−1 layer; placing an nth solution to be printed on the nthscreen, wherein the nth solution comprises one or more of proteins,cells, additional hydrogel, a second constituent, collagen, gelatin, andany combination thereof; pushing a blade across the nth screen to spreadthe nth solution into the exposed nth pattern; and removing the nthscreen to reveal an nth layer positioned on the n−1 layer, wherein n=3,4, 5, 6, 7, 8, 9, 10, or greater.

The appropriate foundation is important to successfully direct the cellsinto the appropriate cell types and structures. For example, referringto FIG. 5, the present inventors have used the PEG-polylysine hydrogelsystem either on its own or in the presence of laminin to support theorganization and differentiation of a range of cell types from neuralstem cells (Hynes et al., 2009; Royce et al., 2007) and endothelialcells in vascular networks (Ford et al., 2006; Li et al., 2006; Rauch etal., 2008; Williams et al., 2012) to retinal progenitors (Hynes et al.,2010; Lavik et al., 2005), retinal ganglion cells, and amacrine cells(Hertz et al., 2013).

The screen printing process increases the survival of cells,particularly those which are sensitive to shear forces and UV-light, byeliminating the shearing associated with more traditional 3D printing,using materials that gel without UV exposure, or using bioinks. The openmatrices lead to robust proliferation and organization of cells, forexample neural or retinal cells. The approach and the materials providedare exceptional for building multilamellar structures, e.g., of theretina or colon, and providing the extracellular matrix cues and supportto foster healthy cells and tissue organization. Preferably, greaterthan 60%, more preferably greater than 70%, and even more preferablygreater than 80% of the cells survive the screening process withoutdifferentiation and without the presence of a matrix which augmentssurvival, e.g., a PEG-based bioink.

One of the major attractions of the process described herein is theability to screen print structures for high throughput screening.Advantageously, 96 or 384 replicates can be printed on a single plate inminutes, making this an extremely efficient method for building uptissue models quickly as well as printing patterns for multiple tissuemodels at the same time. Since printing into small wells is relativelychallenging, layer(s) suitable for imaging can be printed on a substratefollowed by the adherence of PDMS wells or polystyrene colony ringsthereto to create isolated wells.

The tissue model can further comprise a multichannel electrode arraysystem for electroretinography (ERG) measurements or the electrodesrequired for TEER measurement can be patterned into the screen-printedmodel using standard electrical screen printing techniques that arebiologically compatible. The positioning of the electrodes on the tissuemodel will be readily understood by the person skilled in the art.

Advantageously, with the screen printing approach described herein, itis possible to print a layer of cells which will achieve the necessaryguidance to polarize appropriately, when necessary. For example,channels of cell permissive matrix such as laminin-absorbed hydrogelsurrounded by non cell-permissive matrix such as a pure PEG gel serve toorient the cells being printed. By patterning the cell-permissiveprotein gels with non cell-permissive gels, interfaces can be createdthat guide the cell types to polarize in the matrix. The use of theappropriate patterns to polarize cells can be readily determined by theperson skilled in the art.

The screen printing process described herein allows one to build uppatterned, multilamellar systems cheaply and easily with highresolution. All of the materials are readily available and autoclavableincluding the hydrogel components since they are not hydrolyticallydegradable. Screen printing allows one to make many replicates in asingle printing process that preserves the viability of the cellularcomponents and provides the critical extracellular matrix cues to buildretinal structures in three dimensions.

Advantageously, screen printing can complement other methods includingbioprinting to make three-dimensional tissue models. Having multipleapproaches to make tissue models will help to build on the breakthroughswith iPS cells and high throughput screening to understand disease anddevelop treatments. Accordingly, in another aspect of the invention, atissue model comprises (a) multilamellar screen printed layers preparedusing the process of screen printing described herein, wherein greaterthan 60%, more preferably greater than 70%, and even more preferablygreater than 80% of the cells in the screen printed tissue model survivethe screen printing process without differentiation and without thepresence of a matrix which augments survival, and (b) layers positionedusing bioprinting, photolithography, and/or 3D printing. For example,the “substrate” could be an article that was prepared using bioprinting,photolithography, and/or 3D printing, wherein layers are screen printedthereon, as described herein.

The features and advantages of the invention are more fully illustratedby the following non-limiting examples, wherein all parts andpercentages are by weight, unless otherwise expressly stated.

Example 1

Human Optic Nerve Neural Progenitor Cells

We have isolated adult neural progenitor cells (aNPCs) from young andmature optic nerve. These cells are SOX2(+)/Nestin(+)/GFAP(+) (FIG. 2),and can be distinguished from their surroundings by: 1) their ability tobe cultured continuously for >10 replications, 2) their ability to giverise to astrocytes, oligodendrocytes and rarely, neurons, 3) theirability to form neurospheres (FIGS. 3A and B), and 4) theirage-associated depletability. These cells are maintained in a highconcentration matrix medium. Withdrawal of growth medium and replacementwith fetal bovine serum results in differentiation into largely glialcells. Our data suggests that these cells are used during optic nerve(ON) growth in adulthood, to maintain gliogenesis in high stress areas.Loss of these cells results in segmental hypomyelination and ONhypoplasia.

Following aNPC growth and subculture, these cells can be grown ondecellularized ON matrices (FIG. 4A). The human cells grown on thesematrices then “stretch out,” assuming a shape and expression moretypical of cells in vivo (FIGS. 4B and 4C).

The hydrogel screen printing process will allow us to build and identifythe most appropriate artificially constructed extracellular matrix. Byusing an adult neural progenitor cell (aNPC)-artificial matrixconstruct, in conjunction with a retinal ganglion cell (RGC)-survivalassay, we can determine the optimum conditions for aNPC survival,autologous growth factor expression, and RGC-axonal development, allcritical conditions required for normal eye development and growth.

Structure of the Retinal Model

Hydrogel Facilitates Retinal Cell Differential and Organization

The screen printing process avoids the bioprinting shearing aspect whichallows a far broader range of materials and gels to be used. The majorrequirement for screen printing is that the gel be able to set up,crosslink, or phase separate within seconds to a minute, preferably in arange from about 10 seconds to about 10 minutes, more preferably in arange from about 30 seconds to about 5 minutes, without the use of UVlight. This is readily achievable with a range of coupling chemistries.We focus on the vinylsulfone-thiol chemistry because it is easilyperformed and highly biocompatible, avoiding light or toxic couplingagents (Jin et al., 2010; Nah et al., 2002; Zhou et al., 2016).

Polarization of the Cells in the Layers

With the screen printing approach it is possible to print a layer ofcells which will achieve the necessary guidance to polarizeappropriately. For example, vertical channels of permissive matrix suchas laminin-absorbed hydrogel surrounded by non cell-permissive matrixsuch as a pure PEG gel that resists protein absorption and providesstructural guidance cues to orient the cells can be printed. Bypatterning the cell-permissive protein gels with non cell-permissivegels, we are able to create interfaces that will act as architecture toguide the retinal cell types to polarize in the matrix.

Resolution of Features and Reproducibility

The resolution of features in the printing process is dictated primarilyby the resolution of the screens. The 110 mesh screen has pores on theorder of 130 μm. A 200 mesh screen has pores on the order of 74 pun. Inthe electronics industry, resolution down to 20 μm is common (Hyun etal., 2015), but about 100 μm is more than adequate for printing of thetissue models for the purposes of this experiment. Finer meshes can leadto finer resolution features, however, a resolution of about 100 μm ismore than adequate to achieve the polarized orientations in the retina.

Screen Printing Cells

We have tested meshes with 100 micron pores to determine the viabilityof cells post printing. We have used iPS cells differentiated down aneural lineage from a colleague. It was determined that 84+/−2.6% of thecells are viable post-printing in the absence of any survival-augmentingmatrix (Lozano et al., 2015).

Protocol for Screen Printing the Retinal Model for High-ThroughputScreening

Preparing the Cells

Cells are obtained from human donor optic nerves and eyes for which wehave obtained using an UMB IRB exemption, and are currently available inthe lab. These cells are grown and stored as low-subculture passagecultures, and express appropriate markers (Chx10, MBP, GFAP, SOX2,NeuN). Following replating of the subcultures, they are subsequentlydissociated with elastase and placed in the appropriate fluid. Retinalcells are prepared using triturated retinae digested with Papain.Retinal ganglion cells (RGCs) and amacrine cells are isolated followingremoval of microglia using Thy-1 immuno-linked beads (Miltenyi) (Barreset al., 1988; Meyer-Franke et al., 1995).

Photoreceptors are isolated in a similar manner from the Thy-1(−)eluate, but utilizing the appropriate surface markers for photoreceptors(glycosylphosphatidylinositol (GPO)-anchored cell surface moleculeecto-5′-nucleotidase (CD73) for rods). The purified populations (>65%)are then employed in the preparation of cell printing. The RGCs are alsoutilized for co-incubation with the aNPC surface assays. These assaysare prepared in triplicate, and are compared against both commerciallyavailable artificial matrices, as well as laminin-coated surfaces.

Preparing the Screens

The mask for the screen is printed on an inkjet printer on atransparency. The screens are coated with the emulsion, the transparencyis applied, and the screen is exposed to a UV light source followed byrinsing to remove uncrosslinked emulsion. The screens are thenautoclaved and ready for use. Using these standard materialstraditionally used for screen printing t-shirts, we can obtainreproducible features on the order of 100 μm.

Choice of Support Structure for the Retina: Glass Coverslips VersusOxygen Transport Membrane

One can print on a range of substrates including glass coverslips andgas-permeable membranes such as a silicone (polydimethylsiloxane)membrane. For the purposes of the experiments herein, we focus oncoverslips and glass plates because of the ease with which they canfacilitate multiwell culture and high-throughput screening. It isunderstood that the appropriate substrate is not limited to glass platesand coverslips and is readily determined by the person skilled in theart.

Printing Bruch's and ON Membrane Surfaces

A membrane layer that interfaces with the RPE layer consists of laminin,fibronectin, collagen type VI, and glycosaminoglycans (Booij et al.,2010). A cocktail of these molecules can be printed on the foundationalpolylysine-PEG printed gel as a model for the healthy Bruch's membraneand ON surfaces. Alterations to the chemistry of these differentsurfaces can be investigated. A schematic for screen printing a retinalmodel is shown in FIG. 8.

Printing RPE and ON aNPCs

Human RPE and aNPCs cells can be printed on a laminin-coated matrix. Theprinted models can be characterized a number of ways including, but notlimited to, looking at cell survival (live/dead assay), the formation oftight junctions (BESTI; ZO-1) (Brandl et al., 2014; Shadforth et al.,2015), the expression of RPE- and mature glial specific markers (RPE65),MBP, CNPase, Glut-1 (Ahmado et al., 2011), and the response tovariations in Bruch's membrane layer.

Printing Photoreceptors in the Matrix

The photoreceptor progenitors can be printed in the laminin-coatedmatrix. Marker expression as well as phagocytosis of the outer segmentsby the RPE cells can be assessed.

Printing Bipolar, Horizontal, and Amacrine Cells

Different masks can be used to pattern the horizontal, bipolar, andamacrine cells, when present. The bipolar cells can be aligned with thephotoreceptors. The horizontal and amacrine cells will be offset.

Ganglion cells, when present, can be aligned with the bipolar cells inthe laminin-coated matrix.

Culturing the Constructs

The materials completely set within 3 minutes. At that point, theconstructs are immersed in the appropriate media and cultured at 37° C.with media changes twice per week or as needed.

Characterizing the Structures

The constructs are characterized structurally as described belowimmunohistochemically using epifluorescent microscopy, confocalmicroscopy, and a high throughput imager, the acumen cellista. Theacumen cellista uses laser scanning technology using three lasers (blue,green, CyS) to quantitatively assess fluorescent signals. While it cancreate images with a resolution equivalent to 200×, the strength of thesystem is that it can perform a rapid quantification of multiplefluorescent signals through several millimeters of tissue. This willallow us to not only rapidly image many structures but to also quantifythe fluorescence and, therefore, the number of cells and their markerexpression using immunocytochemistry without having to section theconstructs. Synapses in the system will be characterized using anantibody for PSD95 (Schaefer et al., 2016).

Assessing Functionality

One of the attractions of the screen printing process is that it can beused on a range of surfaces. To assess function, the retinal and ONstructures can be printed on multichannel electrode array systems to dothe equivalent of ERG in a dish. This can be coupled with validation bymimicking aspects of diseases, including alterations in Bruch's membranethat are associated with AMD. In addition, the response from differentstructures in response to light exposure can be screened using anLED-based stimulation system (Stett et al., 2003).

Calcification of the elastin layer, crosslinking of the collagen layers,and overall thickening have been associated with a reduction inelasticity that may play a role in AMD progression (Femandez-Godino etal., 2016; Kaluzny et al., 2016). Increases in glycosaminoglycans mayalso play a role in disease (Booij et al., 2010; Fernandez-Godino etal., 2016; Kaluzny et al., 2016). With the PEG-polylysine system as thesupport, these molecules can be varied and the cellular response of theRPE and retinal cells determined.

2-Aminophosphonobutyric acid (AP4), is a blocker of the on-signalpathway in the retina. Advantageously, using the multielectrode arraysystem, the B-wave amplitude before, during and after wash out of thedrug can be measured. The B-wave is primarily due to Muller cell andbipolar cell activity (Stett et al., 2003).

Example 2

With mesh sizes of 100 μm, we obtained the cross piece pattern shown inFIG. 7, having a finest feature of 90 μm wide. The “barbell” of FIG. 7has been designed to promote neural synapses between cell populations inthe narrow region between two cell populations in the square areas.

Meshes with 100 micron or 250 micron pores were also tested to determinethe viability of cells post printing. Since neural-driven iPS cells tendto die or differentiate in response to shear (Faulkner-Jones et al.,2015, Yan et al., 2017), they provide the most robust way to demonstratethe limits on the screen printing technology. Using iPS cellsdifferentiated down a neural linage from a colleague, 84+/−2.6% and93+/−3.5% of the cells were viable post-printing in the absence of anymatrix which typically augments survival for the 100 micron and the 250micron pores, respectively (Lozano et al., 2015). Cell survival wasassessed using the trypan blue exclusion assay. While finer mesh sizesdo impact survival, both are significantly higher than survivalpercentages seen with traditional 3D printers in the absence of bioink.Accordingly, this approach is extremely well suited to building retinaland other tissue structures.

Example 3

An example of a screen printed colon is illustrated in FIG. 9. Thescreen printed colon comprises a neural layer, stromal layer withdendritic cells, and the epithelial/goblet cell layer with the cryptstructures that are critical to normal cell function. The crypts will beprinted by printing gels with holes that are the appropriate dimensions(approximately 200-300 μm) and then printing the epithelial and gobletcell layers.

Although the invention has been variously disclosed herein withreference to illustrative embodiments and features, it will beappreciated that the embodiments and features described hereinabove arenot intended to limit the invention, and that other variations,modifications and other embodiments will suggest themselves to those ofordinary skill in the art, based on the disclosure herein. The inventiontherefore is to be broadly construed, as encompassing all suchvariations, modifications and alternative embodiments within the spiritand scope of the claims hereafter set forth.

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What is claimed is:
 1. A process of making a multilamellar tissue model,said process comprising: (a) positioning a first screen having anexposed first pattern over a substrate; (b) placing a first solution tobe printed onto the first screen, wherein the first solution comprises ahydrogel and optionally an additional constituent; (c) pushing a bladeacross the first screen to spread the first solution into the exposedfirst pattern; (d) removing the first screen to reveal a first layercomprising the hydrogel and optionally the additional constituent; (e)positioning a second screen having an exposed second pattern over thefirst layer; (f) placing a second solution to be printed onto the secondscreen, wherein the second solution comprises one or more of a protein,a cell, additional hydrogel, additional hydrogel comprising anadditional constituent, collagen, gelatin, and any combination thereof;(g) pushing a blade across the second screen to spread the secondsolution into the exposed second pattern; and (h) removing the secondscreen to reveal a second layer positioned on the first layer comprisingthe hydrogel, wherein the process does not require exposure of thelayers to UV light, bioinks, or shearing processes.
 2. The process ofclaim 1, wherein the multilamellar tissue model comprises (i) thesubstrate, (ii) the first layer comprising at least one layer comprisinga hydrogel, and (iii) the second layer comprising one or more of aprotein, a cell, additional hydrogel, additional hydrogel comprising anadditional constituent, collagen, gelatin, and any combination thereof.3. The process of claim 1, wherein the multilamellar tissue modelfurther comprises at least one electrode patterned therein.
 4. Theprocess of claim 1, wherein the model comprises at least onecell-permissive portion and a non cell-permissive portion.
 5. Theprocess of claim 1, wherein the hydrogel comprises poly(ethylene glycol)(PEG), gelatin, or both.
 6. The process of claim 5, wherein the hydrogelfurther comprises an additional constituent selected from the groupconsisting of polylysine (PLL), hyaluronic acid (HA), poly-γ-(glutamicacid) (γ-PGA), poly(aspartic acid) (PAA), poly(arginine), and anycombination thereof.
 7. The process of claim 6, wherein the additionalconstituent further comprises a protein that was absorbed to, or reactedwith, the additional constituent.
 8. The process of claim 1, wherein thesubstrate comprises a material selected from the group consisting ofglass, stainless steel, metal, ceramic, plastic, gas-permeablemembranes, polysiloxanes, fabric, degradable polymer films, degradablepolymer membranes, electrospun materials, and any combination thereof.9. The process of claim 1, wherein the solutions are aqueous and furthercomprise at least one component selected from the group consisting ofphysiologically appropriate buffers, salts, sugars, proteins and drugs.10. The process of claim 1, wherein the second layer comprises cells andwherein greater than 60% of the cells survive the screening processwithout differentiation and without the presence of a matrix whichaugments survival.
 11. The process of claim 1, wherein at least onepattern has a resolution in a range from about 20 μm to about 500 μm.12. The process of claim 1, further comprising the printing of a thirdlayer, said process comprising (i) positioning a third screen having anexposed third pattern over the second layer; (j) placing a thirdsolution to be printed onto the third screen, wherein the third solutioncomprises one or more of proteins, cells, additional hydrogel,additional hydrogel comprising an additional constituent, collagen,gelatin, and any combination thereof; (k) pushing a blade across thethird screen to spread the third solution into the exposed thirdpattern; and (l) removing the third screen to reveal a third layerpositioned on the second layer.
 13. The process of claim 12, furthercomprising printing a fourth layer, optionally a fifth layer, optionallya sixth layer, optionally a seventh layer, optionally an eighth layer,optionally a ninth layer, and optionally a tenth layer using a processanalogous to the printing of any of the first, second, or third layers.14. The process of claim 1, wherein hydrogel layers are able to set up,crosslink, or phase separate without exposure to light.
 15. The processof claim 1, wherein hydrogel layers gel using vinylsulfone-thiolchemistry.
 16. The process of claim 1, wherein the additionalconstituent is selected from the group consisting of polylysine (PLL),hyaluronic add (HA), poly-y-(glutamic add) (y-PGA), poly(aspartic add)(PAA), poly(arginine), and any combination thereof.